Reverse transcription polymerase chain reaction, abbreviated as RT-PCR, is a well known technique for amplifying RNA. In RT-PCR, an RNA strand is reverse transcribed into complementary DNA (cDNA), which is then amplified using DNA polymerase in the polymerase chain reaction. In the first step of this process, cDNA is made from an RNA template using deoxyribonucleotide phosphates and reverse transcriptase together with a DNA primer.
Synthesis of cDNA from the RNA template can be hindered by RNA secondary and tertiary structures, which consist of helices and various other kinds of kinks in the RNA strand. RNA secondary and tertiary structure can be decreased by carrying out the reaction at a higher temperature (e.g., above 50° C.) or by adding denaturing additives. However, the addition of denaturing additives is undesirable because it often reduces reverse transcriptase activity. Higher temperatures also provide the advantage of increasing the specificity of DNA synthesis by decreasing non-specific primer binding. Unfortunately, only a limited number of reverse transcriptases capable of operating at high temperature are currently available, and these exhibit relatively low fidelity DNA polymerization. For example, commercially available Avian Myeloblastosis Virus reverse transcriptase includes RNase H activity and can function at 37° C., but has a fidelity of only about 1.7×10−4. RNase H activity competes with the DNA polymerase activity and the primer binding site and, therefore, cDNA yield is lower. Accordingly, there is a need for reverse transcriptase enzymes that are able to carry out reverse transcription at higher temperatures, including those that have high fidelity and processivity. Such enzymes are beneficial because higher temperatures decrease obstructing RNA secondary and tertiary structure and increase the specificity of reverse transcription by allowing the use of longer and more specific primers.